Electrospray ionization (ESI) is capable of producing intact gas-phase ions from complex biomolecules, and using “native ESI”, even noncovalently bound complexes survive, which could mean that biomolecular ions produced by native ESI are still in their native form in the gas phase. However, structural information of biomolecular ions in the gas phase is difficult to obtain: many methods give either only global information (e.g., ion mobility spectroscopy), or have serious limitations in terms of molecular size (e.g., cryogenic ion spectroscopy). The question whether biomolecular ions produced by native ESI really assume a native-like structure in the gas phase thus has not yet been answered conclusively.

In this lecture, a a new, synergistic approach will be presented, which utilizes ion mobility-mass spectrometry, Förster resonance energy transfer (FRET) of trapped gas-phase ions, and differential ion mobility spectrometry that employs microsolvation by an auxiliary gas, to provide multiple constraints for molecular modeling of gas-phase ion structures. We also developed a novel transition metal FRET method [1] for measuring relatively short distances, 10-40 Å, between a donor dye and a noncovalently bound Cu²⁺ ion serving as a quencher, by measuring fluorescence lifetimes.

Multiple systems are being studied in our laboratory with some or all of the methods mentioned above, including Ala-rich α-helical polypeptides [1, 2], amyloidogenic peptides [3], peptides containing ß hairpins, and so-called “stapled” peptides [4], cyclic peptides with -S–S- bonds; both of the latter fix the gas-phase conformation to some degree. The influence of the length of the molecular linker between the peptide scaffold and the dye moiety is also subject of ongoing investigations. In some cases, seemingly contradictory results were obtained that highlight the problem of relying on only a single method for deriving gas-phase structures. These apparent contradictions could be rationalized by an extensive search of the conformational space of the simulated structural ensemble [2], which identified those that satisfied all the experimentally determined constraints as the most probable structures.

[1] Tiwari, P., Wu, R., J. Metternich, J., and Zenobi, R. Transition Metal Ion FRET in the Gas Phase: A 10-40 Å Range Molecular Ruler for Mass-Selected Biomolecular Ions, J. Am. Chem. Soc. 2021, 143, 11291.
[2] Wu, R. et al., Determining the Gas-Phase Structures of α-helical Peptides: Insights from Shape, Intramolecular Distance, and Microsolvation Assays, Nature Commun. 2023, 14, 2913.
[3] Wu, R., Svingou, D., Metternich, J.B., Benzenberg, L.R., and Zenobi, R. A Transition Metal Ion FRET-based Probe to Study Cu(II)-mediated Amyloid-ß Ligand Binding, J. Am. Chem. Soc. 2024, 146, 2102.
[4] Wu, R. et al., Structural Studies of a Stapled Peptide with Native Ion Mobility- Mass Spectrometry and Transition Metal Ion Förster Resonance Energy Transfer in the Gas Phase, J. Am. Chem. Soc. 2022, 144, 14441.